Fletcher Arritt NC State University Raleigh, NC

Transcription

1 Introduction to Acid, Acidified and Fermented Foods Fletcher Arritt NC State University Raleigh, NC Slide 8-2 Lesson Objectives At end of this module, you will be able to: Identify factors that affect the presence and growth of microorganisms Identify an Acidified Food Identify the characteristics of Clostridium botulinum and the public health concern of C. botulinum and other pathogens in Acid, Acidified and Fermented foods Identify causes for spoilage in Acid, Acidified and Fermented foods 1

2 3 Characteristics and Behavior of Microorganisms All raw foods normally contain microorganisms that will eventually cause spoilage unless they are controlled or destroyed Food preservation is a competition between the human species and microorganisms We attempt to preserve the food that microorganisms attempt to utilize Characteristics and Behavior of Microorganisms Food preservation requires that microorganisms be controlled or destroyed Need to know what they are and how they behave 4 2

3 Characteristics and Behavior of Microorganisms The microorganisms of primary concern to the food processor are molds, yeasts, and bacteria They can grow in food or the processing environment under suitable conditions 5 General Microbiology Helpful bacteria include: Lactic acid bacteria that ferment: Pickles and sauerkraut Cheese and yogurt Helpful yeasts and molds include: Beer and wine yeasts Molds in blue cheese 3

4 Types of Microorganisms Associated With Food Those which can reproduce in the Food Bacteria Fungi ( Yeast & Mold) Those which cannot reproduce in food Viruses Prions Protozoa Slide 8-7 What are top AF concerns? To destroy all microorganisms of public health significance To destroy all microorganisms of non-health significance that could spoil the product under normal conditions of storage and distribution Why does the FDA care if the product is spoiled? Slide 8-8 4

5 Spoilage vs. Illness Spoilage causes changes in a food which make it: Unwholesome Unattractive Unsalable May yaffect quality (BUT NOT NECESSARILY SAFETY) Illness is caused by pathogenic microorganisms and/or their toxic by products Slide 8-9 Where Do Microbes Come From? Ubiquitous in nature (most spoilage, some pathogens) Sources of fecal contamination (mostly pathogens Humans (poor hygiene practices) Slide

6 Where Do Microbes Come From? Waterborne contamination (transport or rinse water) Airborne contamination (dust, moisture) Cross-contamination Contaminated raw product Processing equipment and utensils Slide 8-11 Most bacteria are harmless and many are helpful to humans Coccus or Cocci Bacillus or Bacilli 6

7 Introduction to bacteria Microbes were mostly unknown until the late 1800s, because they are so small Most bacteria DO NOT cause food-borne infections! Some Microorganisms Cause Disease The majority of laboratory-diagnosed d cases of bacterial foodborne illnesses are caused by just a few microorganisms Salmonella spp. Campylobacter Shigella spp. Clostridium perfringens Staphylococcus aureus 14 7

8 Food Poisonings & Infections Food poisoning (intoxication) results from eating a toxin some bacteria produce toxins or poisons when they grow in foods the poison, not the bacteria, causes disease Food infection results from eating live bacteria some bacteria only make you sick when you eat the live cells Clostridium botulinum Gram + sporeforming anaerobe found environmentally How did it get there in the first place? Classified by the type of toxin produced (A, B, C, D, E, F, G) A, B, E are the proteolytic Group I terrestrial est B, E, F are the non-proteolytic Group II marine Slide

9 Clostridium botulinum cont Group I Group II ph 4.6 ph 5.0 aw 0.93 aw 0.97 min. growth temp. min growth temp 10C (50F) 3.3C 3C (38F) D-value 0.2 min Slide 8-17 Clostridium botulinum cont Slide types of illness Infant Wound Foodborne 1. How does it affect humans? 3. Recent examples? 2. What types of products are commonly associated? 4. Other uses? 5. Why do we have a 12D process? 9

10 Other food pathogens Staphylococcus aureus Makes a heat resistant toxin But, unlike C-BOT, Staph does not make spores Salmonella Causes food-borne infections Does not make toxins or spores Escherichia coli O157:H7 Acid resistant Serious food infections Logarithmic Review = =10=1log = = 100 = 2 log 10 3 = 1,000 = 3 log 10 4 = 10,000 = 4 log 10 5 = 100,000 = 5 log 10 6 = 1,000,000 = 6 log Remember you can t subtract exponents 10

11 Exponential (log) growth of bacteria :00 12: :40 8 1:00 Time 16 2: : :00 Number of 2,048 5:00 6:00 Bacteria 16, ,072 1,048,576 7:00 Bacterial Growth Curve Num mber of bacteria a. Lag phase b. Exponential phase c. Stationary phase d. Death phase a b c d 22 Time 11

12 Vegetative cells, Sporeformers, & Toxins Vegetative cells are metabolically active cells and spores exist in dormant state Vegetative cells of yeasts, molds, bacteria, viruses, parasites and heat-labile toxins are easily inactivated by heat Spores and some heat stable toxins can be very heat resistant Examples: Bacillus spores and toxin, Clostridial spores, and Staphylococcal toxin, thermophilic spores Slide 8-23 Heating or cooking foods C-BOT toxin is destroyed Vegetative cells are destroyed However: Staph toxin is heat resistant Spores are heat resistant NOTE: Eating C-BOT spores will not hurt you, but if the spores germinate in foods TROUBLE! 12

13 Microbial Death Curve Microbial death is often a logarithmic i function. This means that the number (but not the proportion) of organisms that die is dependent upon the initial number of organisms present. Population 1e+10 1e+9 1e+8 1e+7 1e+6 1e+5 1e+4 1e+3 1e+2 1e+1 Cell death vs. Time 1e Time (hr) What do we mean by logarithmic cell death? log CFU Cell Death vs. Time Time (min.) Population There exists a 1 log difference in the population between points 1 and 2 min. By calculation: = = /1000 = 90% Death Therefore it takes 1 minute to achieve a 1 log (90%) reduction in bacterial count 13

14 Concept Expanded Exp. Number Log % Tot. % Log red. count # red. red , , , ,000, Initial load 27 Common Examples 4 log g( (4D) reduction in Salmonella for raw almonds (99.99% reduction) 5 log (5D) reduction in Juice pasteurization (99.999%) 12 log g( (12D) reduction in C. botulinum in LACF ( %) One more fun example - Lysol 28 14

15 Where do Spores Come From? Soil Ingredients that originated from or near the soil Fecal contamination Water Environment Slide 8-29 AF: Microorganisms of Concern Pathogens Clostridium botulinum E. coli O157:H7 Bacillus cereus? Staphylococcus h l aureus? Slide 8-30 Spoilage organisms B. stearothermophilus B. coagulans and others C. thermosaccharolyticum C. butyricum Desulfotomaculum l nigrificans Yeasts, Molds and nonsporeforming bacteria 15

16 Factors that Affect Growth Slide 8-31 Atmosphere (Aerobe vs. anaerobe) Temperature (mesophile vs. thermophile) Time ph (acid tolerant sporeforming spoilage organisms not a LACF issue but may be a critical factor) aw (amount of available water, can be a critical factor but usually not an important factor in LACF spoilage and safety) Aerobe vs. Anaerobe Aerobic growth nor microaerophilic growth are of concern in properly canned unopened products Anaerobic growth is a concern due to the preparation of the product steam tunnels, blanching and cappers are designed to remove residual air for a proper vacuum seal Is a properly sealed can anaerobic? What does facultatively anaerobic mean? Slide

17 Mesophile vs. Thermophile Slide 8-33 Psychrotrophs (32-70 F) Capable p of growing g at refrigeration temperatures Mesophiles ( F) Optimum growth at temperatures similar to human and animal body temperatures Most pathogens are in this group Some sporeforming spoilage bacteria Thermophiles ( F) Grow at higher temperatures Problems usually arise in canned foods not cooled, stored or distributed properly Temperature Under normal processing, storage and distribution conditions, if properly retorted, temperature is not a concern However, elevated storage and distribution temperatures may result in thermophilic spoilage Slide

18 Time If delays occur after 1) sealing before processing or 2) in cooling after processing: Growth of spoilage organisms or outgrowth of mesophilic or thermophilic sporeforming spoilage organisms may occur Slide 8-35 Temperature and time abused LACF spoilage concerns If product is held too long before thermally processing, after sealing the container, growth of microorganisms may cause spoilage before the thermal process This type of spoilage is referred to as incipient spoilage Slide

19 Temperature & time abused AF spoilage concerns The aforementioned sporeforming thermophiles may grow in equipment that contacts food if the temperature is within their growth range Microorganisms that grow under these elevated temperatures create spores that are even more resistant to heat Slide 8-37 Temperature & time abused AF spoilage concerns cont. Thermal processes are not designed d to destroy an indefinite number of these spores, therefore. Products and containers should always be held at 170ºF or higher or at room temperature t to prevent the growth of thermophiles Containers should be rapidly cooled below 105ºF after thermal processing as well as stored and distributed below 95ºF Slide

20 Spoilage by Acid-Tolerant Sporeformers Some flat-sour facultative aerobes, such as Bacillus stearothermophilus, are thermophiles Proper cooling after thermal processing and avoiding high temperatures during storage are essential since the thermal process for acid food is not sufficient to destroy their spores 39 Spoilage by Acid-Tolerant Sporeformers Spoilage by the thermophilic anaerobe Clostridium thermosaccharolyticum has been seen in canned tomato products in the ph range 4.1 to 4.5 The thermal process for acidified foods is not adequate to destroy the spores of the organism; however, the problem will not occur if the product is properly cooled and stored at temperatures below 95 F 40 20

21 Temperature Refrigeration slows down food spoilage, but does not stop it! Cold temperatures slow microbial growth, although Some bacteria grow at refrigeration temperatures Listeria monocytogenes Grows at refrigeration temperatures ph and Acidity Most food pathogens don t grow in acid or acidified foods At or below ph 4.6 Clostridium botulinum, (a.k.a. C bot) can NOT PRODUCE deadly botulism toxin. Maintaining proper ph is critically important for acidified foods! 21

22 ph is important! An acidified food with improper ph control can: Appear normal Taste normal ph Requirements In general ph refers to the degree of acidity or alkalinity The ph of a food influences the types of microorganisms that will grow in it In general, yeast and mold grow at a lower ph compared with bacteria All bacteria have an optimum ph range for growth - generally around neutral ph 44 22

23 ph Requirements All bacteria have a minimum below which they will not grow and a maximum above which they cannot grow The ph of foods can be adjusted to help control microbial growth The ph of a food is extremely important with respect to the control of Clostridium botulinum 45 Effect of ph on C. botulinum Growth Spores of C. botulinum and other spoilage types can be found in both acid and low-acid foods In acid or acidified products, the ph is a critical factor for control, with a finished equilibrium ph of 4.6 or less, so that growth and toxin formation will not occur even if the spores of C. botulinum are present 46 23

24 Effect of ph on Required Heat Treatment The application of mild heat destroys all bacteria that are non-sporeformers or all vegetative cells in either low-acid or acid foods, including the vegetative cells of C. botulinum In low-acid foods, high heat must be applied to kill the spores of C. botulinum or the spores of other food spoilage organisms 47 Effect of ph on Required Heat Treatment Thus, these foods must be heat processed under pressure In acid foods, there is no concern with the spores of C. botulinum These spores are prevented from germinating and growing because the ph is 4.6 or below 48 24

25 Effect of ph on Required Heat Treatment Since only the vegetative cells must be destroyed in acid foods, boiling water cooks or hot-fill and hold procedures may be used 49 Lemon Juice Apples Blueberries Sauerkraut Orange Juice Pineapple, canned Apricots Tomatoes, canned Peaches, canned Pears, canned Bananas Beets, canned Asparagus, canned Beef Carrots Peppers, green Papaya 50 Approximate ph Range for Selected Foods Tuna Sweet Potatoes Onions White Potatoes Spinach Beans Peas, canned Corn, canned Soy Beans Mushrooms Clams Salmon Coconut milk Milk Garbanzo Beans Chicken Eggs, whole

26 Spoilage by Acid-Tolerant Sporeformers Acidified d foods do not require a severe thermal process to assure product safety Therefore, a variety of spoilage-causing, acidtolerant sporeformers may survive the process A thermal process for acidified foods is designed to inactivate a certain level of these sporeformers 51 Spoilage by Acid-Tolerant Sporeformers Their survival is typically a result of excessive pre-processing contamination Sometimes underprocessing, either due to an inadequate process or to process deviations, may also result in the survival of these acid- tolerant sporeformers Butyric acid producing anaerobes Aciduric flat-sour sporeformers 52 26

27 Spoilage by Acid-Tolerant Sporeformers The butyric acid producing anaerobes, such as Clostridium butyricum and Clostridium pasteurianum, are mesophilic sporeformers The spores are capable of germination and growth at ph values as low as and consequently are of spoilage significance in acidified foods, particularly if the ph is above Spoilage by Acid-Tolerant Sporeformers Spoilage by butyric acid anaerobes may be controlled either by lowering the ph of the product to below 4.2 or by increasing the thermal process Growth of these organisms in foods is characterized by a butyric odor and the production of large quantities of carbon dioxide and hydrogen gas 54 27

28 Spoilage by Acid-Tolerant Sporeformers Aciduric flat-sours bacteria are facultative anaerobic sporeformers that seldom produce gas in spoiled products The ends of spoiled cans remain flat; hence the term flat sour Spoiled products have an off-flavor fl that t has been described as medicinal or phenolic 55 Spoilage by Acid-Tolerant Sporeformers These organisms (Bacillus coagulans) have caused spoilage in acid foods such as tomato products It is necessary to ensure that the thermal process is adequate to inactivate an expected number of spores The load of flat-sour spores is determined through bacteriological surveys 56 28

29 Spoilage by Acid-Tolerant Sporeformers Pinpointing the ingredient that is contributing the most to the total spore load may prove beneficial in process control For example, proper handling of fruits and vegetables prior to use, such as washing and culling, may also help to reduce spore loads 57 Spoilage by Acid-Tolerant Sporeformers 58 Alicyclobacillus spp., such as A. acidoterrestris and A. acidocaldarius, are flat-sour sporeformers that can grow at a ph as low as 3 in shelf stable juice and other beverage products Spoilage caused by Alicyclobacillus spores has been reported in a variety of juices and beverages (in particular apple juice products), especially when the product packaging allows oxygen transmission 29

30 Spoilage by Acid-Tolerant Sporeformers 59 The spoilage can be minimized by multiple approaches Treating a selected ingredient with an intensified thermal process (at temperatures above 212 F) Product formulation Limiting oxygen availability Rapid cooling of finished products Control of Bacteria by Water Activity For thousands of years people p have dried fruits, meats and vegetables as a method of food preservation It was also discovered that the addition of sugar would allow preservation of foods such as candies and jellies Salt preservation of meat and fish has been practiced over the ages 60 30

31 Control of Bacteria by Water Activity The measure of the availability of water in a food is made by determining the water activity Water activity is usually designated with the symbol a w. 61 Control of Bacteria by Water Activity 62 When substances are dissolved, there is substantial reaction between the substance and the water A number of the molecules of the water are bound by the molecules of the substances dissolved d All of the substances dissolved in the water reduce the number of unattached water molecules 31

32 Control of Bacteria by Water Activity Thus, if some ingredient such as sugar, salt, raisins, dried fruits, etc. is added to food, it competes with the microorganism for available water The water-binding capacity of a particular ingredient influences the amount of water left for the growth of microorganism 63 Minimum a w Requirements for Microorganism Growth Most molds (e.g., Aspergillus) Most yeasts C. botulinum 0.93 Staphylococcus aureus Salmonella some strains some strains Non-sporeforming food-poisoning bacteria readily destroyed by heat

33 Control of Bacteria by Water Activity 65 Examples of foods preserved with this method are Some cheese spreads Peanut butter Honey Syrups Jams and jellies Canned breads Confectionery preparations - toppings Water activity of some common foods Liverwurst 0.96 Cheese Spread 0.95 Caviar 0.92 Fudge Sauce 0.83 Semi-moist Pet Food 0.83 Salami 0.82 Soy Sauce Peanut Butter 15% total moisture 0.70 Dry Milk 8% total moisture

34 Control of Bacteria by Water Activity As far as C. btli botulinum is concerned, a water activity of 0.85 provides a large margin of safety Studies with this organism show that an accurate water activity of 0.93 plus a mild heat treatment will give commercial sterility 67 Regulatory Requirements Related to Water Activity Under the FDA regulation 21 CFR Part 113, a canned food with a water activity greater than 0.85 and a ph greater than 4.6 is considered a low-acid food, and its minimum heat process will have to be filed by the individual packer If reduced water activity is used as an adjunct to the process, the maximum water activity must also be specified 68 34

35 Regulatory Requirements Related to Water Activity If the ph of the product has been adjusted to 4.6 or less and the water activity is greater than 0.85, the product is covered by the acidified food regulation (21 CFR Part 114) and requires only enough heat to destroy vegetative bacterial cells 69 Regulatory Requirements Related to Water Activity Any non-meat containing food, regardless of the ph, with an water activity of 0.85 or less is not covered by the regulations for either the low-acid food (21 CFR Part 113) or the acidified food (21 CFR Part 114) However, these products are covered by FDA s Current Good Manufacturing Practices (CGMPs) regulation (21 CFR Part

36 Methods for Determining a w One commonly used method is an electric hygrometer with a sensor to measure equilibrium relative humidity (ERH) The instrument was actually devised by weathermen, and the sensors are the same as those used to measure relative humidity in air

37 Methods for Determining a w A single measurement of water activity on a food provides information as to which types of microorganisms are most likely to cause spoilage and how close the water activity is to the safety limits 73 Molds Molds are widely distributed in nature, both in the soil and in the dust carried by air Under suitable conditions of moisture, air and temperature, molds will grow on almost any food The black or green discoloration that appears on moldy bread is a common example of mold growth 74 37

38 Molds Molds are also able to survive on a wide variety of substances not normally thought suitable for supporting life These include concentrated solutions of some acids Water containing minute quantities of certain salts Certain pastes used in labeling 75 Molds Mold spoilage of food in closed, processed containers is rare but not impossible Most molds have little heat resistance and cannot survive the thermal processes for lowacid canned foods Therefore, if present, it is the result of serious underprocessing or post-processing contamination Since molds need oxygen to grow, only slight growth can occur unless the food container has an opening to the outside environment 76 38

39 Danger! Improperly Sealed jar lid Air leaks in Aerobic mold grows and raises the ph Anaerobic conditions in the bottom of the Jar C-BOT grows and toxin formed Yeasts Another microorganism of importance to food preservation is yeast Yeasts are single cell microscopic living bodies, usually egg-shaped They are smaller than molds but larger than bacteria 78 39

40 Yeasts Yeasts are widely found in nature and are particularly associated with liquid foods containing sugars and acids They are quite adaptive to adverse conditions such as acidity and dehydration Like molds,,yeasts are more tolerant of cold than of heat 79 Yeasts Most yeast forms are destroyed on heating to 170ºF Spoilage may result from the presence of yeast in canned food, but if this happens, severe underprocessing or leakage must be suspected Usually the growth of yeasts results in the production of alcohol and large amounts of carbon dioxide gas The gas will swell the container 80 40

41 Introduction to Acidified Foods The preservation of foods using acid is older than recorded history y( (ex. yogurt and sauerkraut) The naturally formed acid serves as a preservative for the food and extends its shelflife, but the nutritional quality of the food is relatively unchanged 81 Introduction to Acidified Foods It is not necessary to allow foods to ferment in order to preserve them The same preservative effect can be achieved by adding acids, such as vinegar, to low-acid ingredients, such as vegetables These products are called acidified or acidified low-acid foods 82 41

42 83 Definition of Acidified Foods An "acidified food" is defined by FDA in 21 CFR (b) A low-acid food to which acid(s) or acid food(s) are added to produce a product that has a finished equilibrium ph of 4.6 or below and a water activity greater than 0.85 Examples of acidified foods include: Acidified artichoke hearts, bean salads, peppers or pimentos; Marinated beets or mushrooms; Fresh-pack pickles 84 Definition of Acidified Foods Certain foods have been excluded Carbonated beverages; Jams, jellies and preserves; Acid foods such as dressings and condiment sauces containing small amounts of low-acid food(s) that have a resultant finished product equilibrium ph that does not significantly differ from that of the predominant acid or acid food (sometimes called formulated acid foods ) Fermented foods 42

43 Fermentation Anaerobic catabolism in which an organic compound serves as an electron donor and another serves as an electron acceptor with ATP being produced by substrate level phosphorylation glucose ATP hexokinase ADP glucose-6-phosphate phosphoglucose isomerase fructose-6-phosphate ATP phosphofructokinase ADP fructose-1,6-diphosphate p aldolase dihydroxyacetone phosphate Homofermentative lactic acid bacteria glyceraldehyde-3-phosphate NAD Pi glyceraldehyde-3-phosphate dehydrogenase NADH +H 1,3-diphosphoglycerate ADP 3-phosphoglycerate kinase ATP 3-phosphoglycerate phosphoglyce romutase 2-phosphoglycerate H 2 O enolase phosphoenolpyruvate ADP pyruvate kinase ATP pyruvate lactate dehydrogenase NADH +H pyruvate decarboxylase CO 2 NAD lactic acid acetaldehyde NADH +H alcohol dehydrogenase NAD Ethanol Saccharomyces and similar yeasts 43

44 Summary Two primary types of fermentation organisms Bacteria: Lactic acid bacteria-produce acids Yeasts-produce ethanol Foods are fermented to Preserve Nutrition Uniqueness Sensory properties Economics Three ways to start a fermentation Natural Backslopping Starter culture 44

45 Natural fermentation OD ETOH Yeasts Metschnikowia sp. Pichia sp. Candida sp. Kluveromyces sp. Hanseniaspora sp. Saccharomyces Bacteria acetic acid bacteria lactic acid bacteria Molds Botrytis & others Time Sugar Natural Fermentation Vegetable fermentations Some wines Some cheeses 45

46 Natural Fermentation Positives Distinct flavors Less expensive in the short term Problems Inconsistent end product Limited control of fermentation Scale issues in some industries Backslopping Using a previously successful Using a previously successful fermentation to start the next Still used in brewing industry and in sourdough bread making and small operations Similar positives and negatives to natural fermentation 46

47 Starter cultures Advantages Consistent product Consistent production Increased scale Disadvantage Loss of some exceptional product costs Sauerkraut (Hutkins, 2006) 47

48 Sauerkraut (Hutkins, 2006) Lactic acid bacteria (LAB) Gram (+), non-spore forming, anaerobic, cocci or rods Ubiquitous: plants, animal products, GI tract of man or animals Major role in food fermentation preservation & organoleptic Sugars Lactic acid + other products Exhaust carbon source exhaust nutrients Lower ph out-compete Other products (EtOH, bacteriocins, H 2 O 2 ) antimicrobial Un-stable raw material Stable product 48

49 Two Types of LAB Homofermentative > 85% of products is lactic acid Heterofermentative 50% prod. = lactic acid; 50% others = ethanol, acetic acid, CO 2, The magic of organic acids Dependant on the ph and the pka ph lower than pka most of the acid undissociated [HA] Neutral molecules diffuse into the cell Once inside ph is higher than pka and acid dissociates In an attempt to maintain homeostasis cell exhausts itself and dies 49

50 Spoilage issues Temperature too high or too much salt Inhibits growth of L. mesenteroides May lead to growth of Rhodotorula Causes pinking Temperature too low or salt to low Allow Flavobacterium or Pseudomonas to grow These produce pectinolynic enzymes causing soft kraut L. mesenteroides producing dextrans Add yeast Destem and crush Ferment 65 o F-95 o F Add SO 2 Press Add SO 2 Settle Transfer to new Barrels Age in Barrels Filter Bottle and age 50

51 (Hutkins, 2006) Other fermentations Over 3,500 types consumed Provide % of the worlds food supply Responsible for production of billion dollars worth of food products 51

52 General Guidelines Known organism (preferably LAB) Rapid and continuous ph reduction - below 4.6 in less than 24h Firm should be: (1) evaluating the hazards that could affect food safety based on science not faith (2) specifying what preventive steps, or controls, will be put in place to significantly minimize or prevent the hazards (3) specifying how the facility will monitor these controls to ensure they are working (4) maintaining routine records of the monitoring (5) specifying what actions the facility will take to correct problems that arise. 52

53 Miso Although it's technically done after two months, my ferment takes six months to hit its prime. The longer miso ferments the better it tastes, and as far as I know miso has an indefinite shelf life. I have no knowledge as to when my miso hits a 4.6 ph.. I really know nothing about the ph of miso. QUESTIONS??? 53